Cancer Immunotherapy
Cancer is the second leading cause of death in developed countries. The treatment of certain tumors has improved significantly the last decades. However, conventional cancer therapies such as surgery, radiotherapy and chemotherapy need to be complemented by alternative approaches, particularly for disseminated cancer forms. One promising approach is cancer immunotherapy, which aims at inducing an effective and specific immune response that can control and destroy the cancer cells.
Even if cancer cells often evoke a specific immune response, the response is normally not sufficient to eliminate the malignant cells. This is due to tumor-mediated immunosuppressive mechanisms that block the anti-tumor immune response1. However, these suppressive mechanisms can be reversed by immunotherapeutic strategies aiming at 1) activating professional antigen presenting cells (APC) such as dendritic cells (DC) via e.g. CD40 or Toll-like receptors (TLR), 2) using cytokines, such as IL-2, IL-12 and IFN-α to stimulate the lymphocytes, or 3) blocking signals that suppresses T cell activation by targeting e.g. Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) or inhibitory receptor programmed death 1 (PD-1).
Immunotherapy is already in use for treatment of e.g. bladder carcinoma and malignant melanoma. It has been demonstrated that the cytokine milieu in bladder cancer has an immunosuppressive profile, which makes it a good target for immunotherapeutic intervention2. In fact, intravesical treatment with live, attenuated bacillus Calmette Guerin (BCG) is routinely used for immunotherapy in bladder cancer2. A number of immunotherapies have been tried also for treatment of malignant melanoma. Immunotherapeutic approaches using tumor-infiltrating T lymphocyte infusion in combination with a high-dose of IL-2 have been shown to produce a significant objective response. However, there is a great need for more effective immunotherapeutic strategies for these indications.
The CD40 plays a central role in the immune system, which makes it a highly interesting target for immunotherapy. It is a member of the tumor necrosis factor receptor (TNFR) family and is expressed on a variety of cells in the immune system, such as B cells, monocytes and dendritic cells, whereas the CD40 ligand (CD40L) is mainly expressed on activated T-cells3. CD40L-mediated signalling triggers several biological events, including activation, proliferation, rescue from apoptosis, and production of cytokines and chemokines3. In immunotherapeutic settings, including animal models and clinical trials, anti-CD40 antibodies have been used for direct stimulation of the CD403-6, to block the CD40-CD40L interaction for treatment of autoimmune disorders, or by direct targeting of tumor cells7, 8. Anti-CD40 antibodies have been described as being agonistic, i.e. able to activate cells via the CD40, or being antagonistic, i.e. able to block CD40L induced activation of the CD40. For immunotherapeutic applications aiming at activating the immune response or break tolerance, a strongly agonistic antibody is sought for. Methods that can improve the stimulatory/activating efficacy of such antibody would be highly advantageous.
The CD40 is a type I membrane protein that plays a central role in the immune system. It is a member of the tumor necrosis factor receptor (TNFR) family and is expressed on a variety of cells, e.g. B cells and dendritic cells (DC) as well as several types of carcinoma1;2. The CD40 ligand (CD40L) is mainly expressed on activated Tcells1-7. CD40L-mediated signalling triggers several biologic events, including immune, cell activation, proliferation, and production of cytokines and chemokines (reviewed by1;2). In addition, it can also induce apoptosis in several cancer cells (reviewed by8-10).
It has been shown that the minimal requirement to induce a signal through CD40 is the formation of a receptor dimer11 and that the strength of the receptor mediated signal correlates with increasing valency of an extracellularly applied ligand12. Intracellular signal transmission through CD40 depends on adapter molecules (most notably the TNFR associated factor (TRAF) family) that interact with different intracellular recognition motifs13-15. Extracellular cross-linking of CD40 leads to a stabilisation of these intracellular adaptor molecules, which in turn initiate the signalling cascade.
The effect of CD40 agonists is twofold: i) Immune activation resulting in a tumor specific T cell response, and ii) direct apoptotic effect on CD40 expressing tumors (depending on tumor type). In many studies it has been difficult to separate the mechanisms, but it has been demonstrated that CD40 agonists have anti-tumor effects also on CD40 negative tumors16. CD40 stimulation also has the potential to be used as adjuvant in cancer vaccines.
Pre-clinical studies have demonstrated proof of concept for agonistic anti-CD40 antibody treatment of several cancer types16-21. It has been demonstrated to have an anti-tumor effect on lymphomas, melanoma, hepatoma, osteosarcoma, renal cell carcinoma breast cancer etc. In addition to the potent anti-tumor effect, it has been shown that systemic anti-CD40 treatment also results in side effects (shock syndrome, cytokine release syndrome (CRS)). However, these side effects were not seen when the anti-CD40 antibody was injected directly into the tumor, yet it resulted in systemic anti-tumor effect16. Mice treated intratumorally in one flank were able to clear tumors in the opposite flank16. This anti-tumor effect depends on DC activation and subsequent activation of a CTL response21, which also resulted in a protective immunity to tumor re-challenge. These results have been verified by Jackaman et al22. They studied intratumoral injection of anti-CD40 antibodies alone or in combination with IL-2 in a malignant mesothelioma model (C57BL/6J).
Several humanized or human anti-CD40 antibodies have been evaluated in pre-clinical models23-31. These antibodies have mainly been evaluated in vivo in models based on human tumors xenografted into SCID mice. The effect on CD40 negative tumors has been demonstrated in one study, using SCID repopulated with human monocyte-derived dendritic cells (monocyte-derived dendritic cells) and naïve T cells (Gladue, 2008, ASCO).
Thus, the overwhelming majority of pre-clinical studies using CD40 agonist in cancer therapy have demonstrated a very potent anti-tumor effect. Local, intratumoral CD40 stimulation has been demonstrated to generate a systemic antitumor and metastasis clearing effect, without the side effects associated with systemic CD40 stimulation.
Anti-CD40 treatment has generated promising result in phase I clinical trials, and objective clinical responses have been reported for every anti-CD40 protein drug tested so far10. The CD40 agonists that have been in clinical trials for cancer therapy (recently reviewed by Vonderheide10;32) include CD40L (Avrend™)33, one strong agonistic anti-CD40 antibody (CP-870,893 from Pfizer)34 and one weak agonist (SGN-40, dacetuzumab, Seattle Genetics/Genentech).
Nanoparticles
Nanoparticles, i.e. particles with a diameter typically of approximately 100 to 300 nm, are novel tools with great potential in the field immunotherapy. They can be used as efficient transporters of biomolecules, such as DNA, RNA or proteins either attached to the surface or encapsulated within the particles. Different nanoparticle variants have shown great potential, both for sustained release of carried biomolecules and for activation of antigen presenting cells for induction of a cytotoxic T lymphocyte (CTL) response10. Several different particle types are currently evaluated in nanomedical applications, such as polystyrene particles, poly (lactic-co-glycolic acid) (PLGA) particles and poly (γ-glutamic acid) (γ-PGA) particles. Polystyrene particles have been shown to be effective in generating DC maturation11, however, they are not biodegradable and therefore less suitable for therapeutic applications. PLGA particles on the other hand are fully biodegradable, however, there are problems associated with this method, such as low encapsulation efficiency of water-soluble proteins and instability arising during the formulation, storage and lyophilization of the nanoparticles. Recently, a novel protein delivery system has been described based on self-assembled amphiphilic polymeric γ-PGA nanoparticles, which are fully biodegradable36. It has been demonstrated that these particles are efficient for protein delivery and can be used to induce a specific T cell response in a vaccine model system10.
Slow release compositions comprising an anti-CD40 antibody encapsulated in dextran-based particles, for use in cancer treatment, have also been reported45. The dextran particles used were in the microscale-range and were constructed to encapsulate the antibodies within the particles. The objectives with such slow-release formulations in local administration approaches are several, including minimization of antibody leakage from the local area to be treated, in order to reduce toxicity and other adverse effects associated with the systemic release of the antibody. In addition, slow-release may achieve reduced dosing frequency.
Despite the above developments in the fields of immunotherapy and nanoparticle use, there remains a need for new therapies for stimulating the immune system, in particular in the treatment of cancer.